Nu-beta, gamma-dihydroxy and diacetoxy-2, 4-dinitropyrroles



United States Patent 3,249,624 N-BETA,GAMMA-DIHYDROXY AND DIACETOXY- 2,4-DINITROPYRROLES George Karmas, Bound Brook, N.J., assignor to Ortho Pharmaceutical Corporation, a corporation of New Jersey No Drawing. Original application Nov. 5, 1964, Ser. No. 409,277. Divided and this application Sept. 7, 1965,

Ser. No. 485,546

3 Claims. (Cl. 260-3263) This is a division of application Serial No. 409,277, filed November 5, 1964, which is a continuation-in-part of my co-pending application Serial No. 23,383, filed April 20, 1960 (now abandoned), and application Serial No. 210,199, filed July 16, 1962, now abandoned.

The present invention relates to l-substituted-2,4-

in which R is a substituent selected from the group con s isting of alkyl groups selected from the group consisting of n-propyl, n-butyl, iso-butyl, sec-butyl, n-amyl, iso-pentyl, sec-pentyl and n-hexyl, haloalkyl groups selected from the group consisting of 'y-bromopropyl and Zi-bromobutyl, acetonyl, ,6,-'y-dihydroxypropyl, p,' -diacetoxypropyl, l-(N-morpholino carbonyl methyl), piperazino groups selected from the group consisting of 1- (N -methyl-N -piperazino carbonyl methyl) and l-(N acetyl-N -piperazino carbonyl methyl), and 1-(2-methyl- S-nitroimidazolyll-ethyl) Examples of 2,4-dinitropyrroles coming within the scope of the above formula are: 1-('y-bromopropyl)- 2,4-dinitropyrrole, 1-acetonyl-2,4-dinitropyrrole, l-isopentyl-2,4 dinitropyrrole, 1 (figy-dihydroxypropyl)-2,4- dinitropyrrole and l-(N-morpholino carbonyl methyl)- 2,4-dinitropyrrole.

The compounds of the present invention have antimicrobial activity and more specifically are effective against T richamonas foetus, a parasitic protozoan that infects the uterus of animals and causes abortion in cattle. The high order of trichomonodicidal activity possessed by .the compounds of the present invention is quite surprising as the closely related 1-alkyl-2,5-dinitropyrroles and l-alkyl-3,4-dinitropyrroles have no useful activity.

It is an object of the present invention to provide new compounds having therapeutic utility in the treatment of animals infected with Trichomonas foetus.

It is also an object of this invention to provide a new method for the preparation of 1-substituted 2,4-dinitropyrroles.

Heretofore it has been proposed to prepare N-alkylpyrroles by reacting pyrrole with potassium metal in an organic solvent such as a low boiling hydrocarbon and Patented May 3, 1966 reacting the resulting N-potassiumpyrrole with an alkyl halide, for example, methyl iodide. The latter reaction is carried out either by heating the ingredients together in a sealed tube at 130 C., or by refluxing in a low boiling organic solvent. By this method, N-methylpyrrole can be obtained in yields around 25-50%. A considerable amount of pyrrole is converted to a-methylpyrrole, which side reaction is primarily responsible for the relatively low yield of the desired N-alkyl product.

It has also been proposed to react an alkali metal pyrrole in anhydrous liquid ammonia with an organic halide. Under such conditions, high yields have been obtained, but the anhydrous liquid ammonia solvent is more difiicult to handle than a solvent having a lower vapor pressure. Moreover, the alkylation reaction, at liquid ammonia temperature, requires several hours for completion.

In accordance with the present invention, the alkylation of 2,4-dinitropyrroles is effected in dimethylformamide which is a superior solvent for this reaction. In general, the alkali metal salt of 2,4?dinitropyrrole and an excess of the alkylating agent, which may be a chloride, bromide, iodide, sulfate, sulfonate, etc., are heated in dimethylformamide for varying periods of time (determined by the reactivity of the alkylating agent). The reaction product is isolated and purified by conventional procedures of extraction, distillation, recrystallization, etc. In those instances where the grouping introduced at the 1-position of the pyrrole contains a functional group, further reactions may be performed on this group.- For example, an ester may be hydrolyzed to a carboxylic acid and the latter may further be converted to an acid chloride, from which a wide variety of derivatives may be prepared.

The following examples will serve to illustrate more fully the method of preparing the novel compounds of the present invention.

EXAMPLE I 1 -buzyl-2 ,4 -dinitr0 pyrrole A mixture of 5 grams (0.0 275 mole) of thesodium salt of 2,4-dinitropyrrole, 8 milliliters of dibutyl sulfate, and 20 milliliters of dimethylformarnide is heated to a gentle boil and after one minute is cooled and poured with stirring into a mixture of 200 milliliters of water, 100 grams ice, and 15 grams of sodium carbonate layered with 150 milliliters of ether. This mixture is shaken vigorously and then the layers are separated. The ether solution is washed with 100 milliliters of 5% aqueous sodium carbonate, dried with anhydrous magnesium sulfate, concentrated and then distilled. The l-butyl-2,4-dinitropyrrole is a pale yellow oil which dist-ills .at l35 C. at 0.2

Unalkylated 2,4-dinitropyrrole may be recovered from the aqueous sodium carbonate solutions by acidification and extraction with ether.

Dimethyl sulfate, diethyl sulfate, and dipropyl sulfate have also been reacted with the sodium salt of 2,4-dinitro pyrrole following this procedure.

3 EXAMPLE II 1 -isamyl-2,4-dinitropy rrole A mixture of 5 grams (0.0275 mole) of the sodium salt of 2,4-dinitropyrrole, 6 milliliters of isoamyl bromide, and 10 milliliters of dimethylformarnide is heated under reflux for two hours. The reaction mixture is worked up as described for the l-butyl analog (Example I), and distillation affords 1-isoamyl-2,4-dinitropyrrole as a pale yellow oil which distills at 135-140 C. at 0.1 mm.

EXAMPLE III grams (47.2% yield) of pale yellow prisms, melting point From the aqueous sodium carbonate solutions, a 35% recovery of 2,4-dinitropyrrole is effected an acidification with hydrochloric acid and extraction with ether.

EXAMPLE IV 1-(2,3-dihydr0vcypr0pyl) -2,4-dinilr0pyrr0le A mixture of 5 grams (0.0275 mole) of the sodium salt of 2,4-dinitropyrr-ole, 6 milliliters of a-glyceryl monochlorohydrin, and milliliters of dimethylformamide is heated under reflux for fifty minutes, cooled slightly, and then concentrated under vacuum to remove the solvent. The viscous concentration residue is leached with three BOO-milliliters portions of .boiling ether, decanting each time from the insoluble residue. The combined ether solution is concentrated and the oily residue is distilled to afford the crude product as a viscous oil which boils at 200-210 C., at 0.01-0.02 mm. It solidifies on standing and is recrystallized from ethyl acetate to give 2.4 grams (37.8% yield) of 1-(2,3dihydroxypropyl)-2,4-dinitropyrrole, small cream prisms of melting point 111-112 C.

This procedure is generally suitable for the preparation of water soluble 1-substituted-2,4-dinitropyrroles because it avoids the loss of these in aqueous wash solutions. It has been applied to the synthesis of the corresponding 1- (fi-hydroxyethyl) and l-( y-hydroxypropyl) analogs.

EXAMPLE V 1 -(l3,'y-diace ioxypropyl -2,4-dinilr0pyrr0 [e A mixture of 7.3 grams of 1-(fl,'y-dihydroxypropyl)-2,4- dinitropyrrole (Example IV) and 60 milliliters of acetic anhydride is heated under reflux for ninety minutes, cooled slightly, and then concentrated under vacuum to remove the excess of acetic anhydride. The syrupy residue is dissolved in 20 milliliters of boiling ethyl acetate and the solution is stored at 0 to crystallize 8.4 grams of pale yellow granules which melt at 109 110. A second crop of 1.0 gram is isolated by concentration of the mother liquor. The total of 9.4 grams represents a yield of 94%.

EXAMPLE VI 1 -carb oxymethy l-2,4-dinitr0pyrr0 le To a cold (5 C.) solution of 5.2 grams (0.0214 mole) of 1-carbethoxymethyl-2,4-dinitropyrrole (prepared according to the process of Example II, from ethyl chloroacetate) in 150 milliliters of methanol is added 0.855

gram (0.0214 mole) of sodium hydroxide in 10 milliliters of water. To the resulting paste is added 200 milliliters of water and this mixture is warmed at 45 C. for 10 minutes. After it has been cooled to 0 C., this solution is acidified with hydrochloric acid and the precipitated carboxylic acid is filtered off, washed on the filter with cold water, and dried in 'air. The 1-carboxymethyl-2,4-dinitropyrrole thus isolated, in almost quantitative yield, is hydrated. It melts at 205-207 C., after slow loss of water of hydration on the melting point stage. Q

Calcd. for C H O N C, 33.50; H, 2.34. Found: C, 33.63; H, 2.60.

EXAMPLE VII '1-(4-methylpiperazinocarbonylmethyl)- 2,4-dinitropyrrole A mixture of 10 grams of 1-carboxymethyl-2,4-dinitropyrrole (Example VI) and 75 milliliters of thionyl chloride is boiled under reflux for two hours and then concentrated under vacuum at 60 C. The dark oily residue is dissolved in 50 milliliters of toluene and reconcentrated under vacuum at 60 C. The residue is dissolved in milliliters of methylene chloride and this solution is stirred at 0 while a solution of-S grams of N-methyl-piperazine in 40 milliliters of methylene chloride is added over ten minutes. After being allowed to stand at 25 for fifteen hours, the reaction mixture is shaken with 20 milliliters of 10% aque-oussodium carbonate and the methylene chloride layer is separated and dried with magnesium sulfate and then concentrated to a solid residue. This is recrystallized from ethyl acetate to afford 8.4 grams (61%) of 1-(4-methylpiperazinocarbonylmethyl)-2,4-dinitropyrrole as buff flakes which melt at 121.

EXAMPLE VIII 1-(N-pyrrolidocarbonylmethyl) -2,4-dinitr0pyrr0le A mixture of 10 grams of the sodium salt of 2,4-dinitropyrrole, 9.4 grams of N-chloroacetyl-pyrrolidine, and 40 milliliters of dimethylformamide is boiled under reflux for fifteen minutes and then poured into 600 milliliters of cold 5% aqueous potassium carbonate. The insoluble solid is filtered ofi, dried in air and decolorized with charcoal in an acetone solution. The clear filtrate is evaporated to dryness and the solid residue is recrystallized from ethyl acetate to afford 12 grams (80%) of 1- (N-pyrrolidinocarbonylmethyl)-2,4-dinitropyrrole as pale yellow prisms which melt at 158159.

EXAMPLE 1 -(,B-br0m0ethyl) -2-methyl-5-nitr0imidazole Fifty milliliters of thionyl bromide isstirred vigorously and maintained at 50-60 while a total of 47.5 g. of l-(fi-hydroxyethyl) 2 methyl-S-nitroimidalole is added over a period of fifteen minutes. The reaction mixture is then maintained at 80-85 for fifteen minutes, cooled slightly, and poured onto ice, the last of the contents being rinsed from the reaction vessel with methylene chloride and a little water. Three hundred milliliters of methylene chloride is added to the hydrolysis mixture which is then stirred and maintained at 5 while enough solid potassium carbonate is added to attain a permanent slightly alkaline pH. The mixture is now filtered and the layers of the filtrate are separated. The methylene chloride portion is dried with magnesium sulfate and concentrated under vacuum to a solid yellow residue potassium salt of 2,4-dinitropyrrole.

Pale yellow granules which melt at 81-82 may beobtained by recrystallization from ether.

EXAMPLE X compounds of the present invention prepared according 1-[(2,4-dinitropyrryl-1)ethyl]-2-me-th l-5 to the methods illustrated in Examples I through X above,

appear in Tables I, II and III. The constants of commlxture of 17 grams of the potasslul'n'sa1t of pounds not listed in Table III may be found in the dedinitropyrrole, 20 grams of 1-(B-bromoethyl)-2-methyl-5- 5 tailed Examples I through X nitroirnidazole, and 70 milliliters of dimethylformamide is stirred and boiled under reflux for fifteen minutes and TABLE Y fhE2E FROM then poured into one liter of cold 5% aqueous potassium carbonate. The insoluble solid is filtered off, dried in air ON and decolorized with charcoal in an acetone solution. J The pale orange acetone filtrate (about 600 milliliters in p volume) is boiled down to a volume of 150 milliliters I and chilled at 0 to effect crystallization of 16.4 grams of 1-[(2,4 dinitropyrryl-l)-ethyl]-2-methyl-5-nitroimidazole, yellow granules which melt at 186187. R a iir rgt iryi i i ic lel Yleld, percent A second crop of 2.1 grams is obtained on further concentration and chilling of the mother liquor, bringing 1 min at the total to 18.5 grams, a yield of 4 83 The experimental details and physical contents of other TABLE II.-ALKYL-2,4-DINITROPYRROLES, FROM ALKYL HALIDES 2N'iv NJNO2 R R Alkylating Agent Reaction Conditions Yield,

- (in Dimethylformampercent ide) n-C H1 11-03mm 3% hrs. at l 47 is0-C H9 iso-C HpBr 64 sec-0M sec-CJInBr 30 D-C5I'I11 l'l-OSHHBI 77 130-0511 lso-ca uBl 74 sec-CEBU, SEC-C5H11BI 2g n-Oe l3 fl rsBl i 74 CI'I2CH2OH2BI I BICHZCHZOIIQBI 54 CH COCH; CH3 OCH CI 78 CHZCHOHCHZOH OHZOIIGIIOI'IOHZCI 39 OH2CH2CH2CH2BI' BICHZCHZCHZCIIQBI 10 min. at 66 OHgHCHgO C 0 CH3 (Note 1) (Note 2) 94 01120112 /OH CH' CI'IgOON' O- ClCHzC ON\ 0' 15 min. at 75 CHgCIIz 0112-062 CH CI-I CHZC ON NOH3 (Note 3) (Note 4) 61 p a e V CHZC ON NC 0 CH3 (Note 5) 1 (Note 6) 25 0112011241: N 1 BICHECHFDI N 15 at 70 Note 1: By acetylation of R=CH CH-0H CH O-H.

Note 2: 1% hours at 140 in excess acetic'anhydride.

Note 6: A hour at 5 in methylene chloride.

TABLE IIL-PHYSICAL CONSTANTS- OF l-ALKYL-2,4-DINITROPYRROLES OF TABLES I AND H O2N'I LN/NOZ I R Y Analytical R 13.1. 0.1mm. M.P.,

degrees Oalcd. Found n-CaHv 125-130/.2 42. 21 4. 55 11.. 42.26 O;4.65H 11-04119 130-135/. 2 45. 07 0; 5. 20 H 45. 02 O; 5. 39 H is0-C4H9 137-129/. 4 45. 07 0; 5. 20 H 45.31 0; 5.18 H sec-C4110 125-130 1 45. 07 0; 5. 20 H 45. 49 C; 5. 27 H n-CH11 146-148/. 2 18. 49 N 18. 62 N. is0-C H 1 135-140/. 1 47. 47 O; 5. 77 H- 47. 65 o; 5. 72H sec-C511 110-115/. 005 47. 57 C; 5. 77 46. 87 C; 5. 75 H n- 6H! 125-130/. 002 49. 78 C; 6. 27H--- 49. 71 0; 6. H CHgCHgCHzBr 165170/. 01 30. 43 C; 2. 90 H 30. 23 O; 3. H CH CI-I CH CHzBt ISO-185]. 003 32.88 C; 3. H 33. 28 o; 3. 62 H CHzCOCHa 39.44 O;3.31H 39.88 C;3.35H CHZCHOHCHZOH 200-210/.o2 36.37 0; 3. 92H.-- 36.16 C; 3. 03 H CHzCHCH OCOCH; 109-110 41.91 O;4. 16 H 42.16 C;4.00 H.

CH CI-I CHzCON\ 0 145-146 42.25 0; 4. 26H--. 41.01 O;4.15 H.

CHzCg CHZCHB CHzOON N-CH; 120-121 44. 44 C; 5.09 H 44.48 C;5.16H.

CHgCHz CHgCHg CHZCON V N-COCHS 215-216 44.31 c; 4. 65 H 44.09 C; 4. 68H.

CHZCHB CHZCHPN N 186-187 38.71 C; 3. 25 H- 39.09 O;3. 19 H.

The in vitro trichomonadicidal activity of the comwhich no viable organisms are present at the ninth day pounds of the present invention may be demonstrated by examination. If there are no viable organisms present on a series of tests which establishes the minimal inhibitory examination at a time less than nine days, the concentraconcentration of these compounds. Minimal inhibitory tion ofcompound is greater than minimal and if there are concentration, as used above, is defined as the minimal viable organisms present at the ninth day examination, concentration ofa trichomonadicidal compound capable the concentration is less than minimal. The results of of preventing the growth of and killing T richomonas these tests appear in column 1 of Table IV, wherein the foetus organisms introduced into a culture medium, caconcentration of the 1-substituted-2,4-dinitropyrrole-s is pable alone of supporting a vigorous growth of the oreXpl'essed 111 pa ts per million. ganisms and containing the trichomonadicidal compound The tOXiCltY 0f compounds of the Present invention t be te t d. Th cult e di m u d i th {tests i are determmed by oral administration to mice and may be described in a publication of Kupferberg, Johnson and. determined r m e in vivo data appearing in Table IV. Sprince, Proceedings of the Society for Experimental Bi- In column 11nd the heading 50 is indicated the ology and Medicine, volume 67, pages 304-308, 1948. q a y mllhgrams p kilogram of y weight) In making the tests to determine minimal inhibitory fatal to 0f t e mice tested. The amount concentrations, 0.05 milliliter of a 48-hour culture of. mllhgrams P kilogram of y Weight) required to Trichomvnas foetus is placed in -a series of tubes containcure 0f t e t a mals injec ed With a lethal dose mg 10 lmilliliters of the culture medium and increasing of Trichomonas foetus is indicated in column 3, under the amounts of the compound to be tested. The inoculated heading PD In this column, NP. indicates no proculture medium is then incubated at 37 C. for nine days tection. Column 4 of Table IV reports the therapeutic and examined under magnification after two, five, seven, index of these compounds. A therapeutic index of at and nine days. The minimal inhibitory concentration of least 10 is preferred by pharmacologists as providing a the compound tested is that concentration in the tube in sufficient margin of safety between the LD and the PD TABLE IV.TRICHOMONADICIDAL ACTIVITY OF 1-SUBSTITUTED-2,4- DINITROPYRROLES In vivo R In vitro activity,

ppm. LD50 PD50 T.I

moire 100 000 30 30.0 11-C4H9 100. 1, 200 53 23. 0 is0-C I-I0 10. 0 1, 000 84 12. 0 sec-0.115 10.0 1, 200-1, 250 45 33.0 11-05111 20. 0 1, 500 50 30 1so-O5H11 100 1, 100 5. 3 200.0 sec-C 11 20. 0 2, 150 100 21. 5 n-G H 10. 0 2, 150 140150 14. 3 CIIzCHzCHzBI 10. 0 1, 400 120 12. 0 OHZCHBCHZOmBr 0.4 1, 000-2, 000 75-100 -20 0112000133 10.0 1,000 37.5 27.0 CHzCHOHCHaOH 2.0 1, 050 1520 52.0

OOOCH3 CH CHOH OCOCHa 10.0 1,900 -30 76 CH CH C1120 O-N 0 1. 0 1, 500 5-7 200 CH 2CH;

0112c O-N 10 011;. 10.0 1,050 10.5 100 CH CH CHzC O-N NC 0 CH3 10. 0 1, 050 67-135 10. 5

CH CH GHQCHFN N (Inactive) 3, 000 10.5 300 In employing the tric-homonacides of the present in- EXAMPLE XII vention for the treatment of T richomonas foetus, one or Deionized water 9030 more of the active agents are uniformly distributed in a Methyl cellulose 350 suitable chemotherapeutic vehicle that is chemically com- Glycerin 500 patible with the particular trichornonacide selected, nonpara hydl oxy benzoic acid methyl ester 0'20 inhibiting with respect to the action of the effective agent 1 (5 hydroXypmpy1) 2 4 dinitmpyrro1 LOO upon Trichomonas foetus and essentially non-injurious to the vaginal mucosa under the conditions of use. The ve- 5 XA P XIII cle is preferably of a liquid or semi-liquid type. Fur- Deionizgd Water 8080 t ermore, since the final preparation should be readily Pectin 800 c ble With vaginal fluids, the vehicles, whether hydrous Propylene glycol 10.00 or anhydrous, are preferably Water-miscible or water-dispara hydroxy benzoic acid methyl gster 0.10 perslble. The compositions of this invention may be in pal-a hydroxy benzoic acid propyl ester the form of supposltones, 1f deslred. a hydroxybutyl) 2,4 dinitropyrmle 1.00

The foregoing criteria for a vehicle in which the compounds of the present invention are incorporated may be met by a large number of semi-liquid chemotherapeutic vehicles that are well known in the art. Thus, for example, the vehicle may comprise semi-liquids that are colloidal in nature, especially those that are viscous and/ or mucilaginous in character. Such vehicles are particularly suitable for use in topical treatment of Trichomonas foetus because of their inherent gelatinous and miscible nature which afiords prolonged contact between the l-substituted- 2,4-dinitropyrrole and the infecting organism.

In order to disclose more clearly the manner of formulating the compounds of the present invention to topical application, several specific examples will hereinafter be described in considerable detail.

EXAMPLE XI Deionized water 75.80 Sodium carboxymethyl cellulose 3.00 Polyethyleneglycol (molecular weight approximately 4000) 15.00 Propylene glycol 5.00 Para-hydroxy-benzoic acid methyl ester 0.20 1-methyl-2,4-dinitropyrrole 1.00

The trichomonadicidal formulations of Examples XI through XIII are prepared according to the following general procedure in which two initial solutions are mixed to make the formulation, all the parts being given by weight. To prepare Solution A, dissolve the para-hydroxy-benzoic acid methyl ester in about two-thirds of the hot deionized Water, 'cool to about F., and, While stirring, add the gel-forming ingredient and glycerine or propylene glycol. To prepare Solution B, add the trichomonadicidal agent to the remainder of the deionized water, and adjust the pH to the desired value. The formulation is prepared by adding Solution B to Solution A in a slow stream with good stirring; stirring is continued for at least one hour.

Certain compounds of the present invention have also been found to be effective against enterohepatitis (blackhead) when administered by admixture, suspension, or dispersion in the food and/or drink normally partaken by turkeys, such as grain, mash, scratch, water 01' other liquids.

The general range of concentration of the l-substit-uted- 2,4-dinitropyrrole in the total substance is from about 0.05% or less to about 1%. The optimal concentration 11 for effective therapy is in the range from about 0.05% to about 0.2% of the total food or drinking Water. With these optimal concentrations, the daily drug intake for infected birds varies from about 20 milligrams of drug per kilogram of body weight to about 400 milligrams of drug per kilogram of body weight. In general, the precise dosage depends on the particular compound and the severity of the infection. Many of the compounds of the present invention may be administered in the concentrations indicated above with little or no toxic eflect.

Various changes and modifications of the invention may be made and to the extent that such variations incorporate the spirit of this invention, they are intended to be included within the scope of the appended claims.

What is claimed is:

1. A compound of the formula 2. A compound of the formula 0 rN-U 5 L'LNO:

omonorromon 10 3. A compound of the formula No references cited.

HENRY R.

JILES, Acting Primary Examiner. 

1. A COMPOUND OF THE FORMULA 